It takes two: Building the vertebrate skull from chondrocranium and dermatocranium

Abstract

In most modern bony vertebrates, a considerable portion of the chondrocranium remains cartilaginous only during a relatively small window of embryonic development, making it difficult to study this complex structure. Yet, the transient nature of some chondrocranial elements is precisely why it is so intriguing. Since the chondrocranium has never been lost in any vertebrate, its function is critical to craniofacial development, disease, and evolution. Experimental evidence for the various roles of the chondrocranium is limited, and though snapshots of chondrocranial development in various species at isolated time points are valuable and informative, these cannot provide the data needed to determine the functions of the chondrocranium, or its relationship to the dermatocranium in evolution, in development, or in disease. Observations of the spatiotemporal associations of chondrocranial cartilage, cartilage bone, and dermal bone over early developmental time are available for many vertebrate species and these observations represent the data from which we can build hypotheses. The testing of those hypotheses requires precise control of specific variables like developmental time and molecular signaling that can only be accomplished in a laboratory setting. Here, we employ recent advances in contrast-enhanced micro computed tomography to provide novel 3D reconstructions of the embryonic chondrocranium in relation to forming dermal and cartilage bones in laboratory mice across three embryonic days (E13.5, E14.5, and E15.5). Our observations provide support for the established hypothesis that the vertebrate dermal (exo-) skeleton and endoskeleton evolved as distinct structures and remain distinct. Additionally, we identify spatiotemporal patterning in the development of the lateral wall, roof, and braincase floor of the chondrocranium and the initial mineralization and growth of the bones associated with these cartilages that provides support for the hypothesis that the chondrocranium serves as a scaffold for developing dermatocranial bones. The experimental protocols described and data presented provide tools for further experimental work on chondrocranial development.

Keywords: Cartilage; cartilage bone; dermal bone; skull evolution.

Fig. 1.

Major events in the evolution of cranial cartilage and bone. The phylogeny of these vertebrate clades is based on Janvier (2015) and Brazeau & Friedman (2015). Figures of cyclostomes, osteostracans, and placoderms are adapted from Romer (1959). * indicates a paraphyletic group; ^† denotes extinct groups.

Fig. 2.

Mouse embryos aged chronologically at E12.5 (A) and E13.5 (B, C) stained using alcian blue (glycosaminoglycans in cartilage) and alizarin red (calcium in bone) and then optically cleared showing the braincase floor of the early chondrocranium at E12.5 (A) and the lack of staining of cranial bones at E13.5 (B & C). Inferior view (B) and lateral views (A & C) with identification of chondrocranial features. The globe of the eye blocks most of the ala orbitalis in the lateral view at E13.5 (C). Abbreviations: AO, ala orbitalis; AR, acrochordal cartilage; COP, orbitoparietal commissure; CSE, sphenethmoid commissure; H, hypophyseal cartilage; OA, occipital arch; P, parachordal cartilage; PCA, pars canalicularis; PN, paries nasi; PP, parietal plate; SN, septum nasi; T, trabecular cartilage; TT, tectum transversum; Y, hypochiasmatic cartilage.

Fig. 3.

3D reconstruction of PTA-enhanced μCT images of the head of a mouse harvested at E13.5 showing the lateral roof, lateral wall, and braincase floor of the chondrocranium. Only the left side is fully segmented and the otic and nasal capsules are not included. Using the eMOSS staging system (Musy et al., 2018), this mouse is staged at 342.5 ± 2 hours or approximately 14.25 days post conception, revealing a developmental age that is older than its harvesting age. A) Lateral view of segmented regions within 3D volume rendering of transparent head to show relative placement of the chondrocranium; B) Lateral view of segmented regions of the chondrocranium and identification of chondrocranial features; C) Superior view of segmented regions within 3D volume rendering of transparent head to show relative placement of the chondrocranium within the head (the right side was not completely segmented); D) Superior view of segmented regions and identification of chondrocranial features. Scale bar on the left corresponds with A and C; scalebar on right corresponds with B and D. Abbreviations: AO, ala orbitalis; AR, acrochordal cartilage; fhg, foramen hypoglossum; H, hypophyseal cartilage; OA, occipital arch; P, parachordal cartilage; PP, parietal plate; T, trabecular cartilage; TP, tectum posterius; TTR, tectum transversum; Y, hypochiasmatic cartilage.

Fig. 4.

3D reconstruction of PTA-enhanced μCT images of the head of a mouse harvested at E14.5 showing the lateral roof and lateral wall and braincase floor of the chondrocranium. Only the left side is fully segmented and the otic and nasal capsules are not included. This mouse is developmentally staged (Musy et al., 2018) at 368 ± 2 hours, or E15.3 days post conception, revealing a developmental age that is older than its harvesting age. A) Lateral view of segmented regions within 3D volume rendering of transparent head to show relative placement of the chondrocranium; B) Lateral view of segmented regions of the chondrocranium and identification of chondrocranial features; C) Superior view of segmented regions within 3D volume rendering of transparent head to show relative placement of the chondrocranium within the head (the right side was not completely segmented); D) Superior view of segmented regions and identification of chondrocranial features. Scale bar on the left corresponds with A and C; scalebar on right corresponds with B and D. Abbreviations: AO, ala orbitalis; AR, acrochordal cartilage; COP, orbitoparietal commissure; CSC, sphenocochlear commissure; CSE, sphenethmoid commissure; fhy, hypophyseal fenestra, fhg, foramen hypoglossum; H, hypophyseal cartilage; O, orbital cartilage; OA, occipital arch; P, parachordal cartilage; PP, parietal plate; T, trabecular cartilage; TP, tectum posterius; TTR, tectum transversum; Y, hypochiasmatic cartilage.

↑ Fig. 5.

3D reconstruction of PTA-enhanced computed tomography images of the head of a mouse harvested at E15.5 showing lateral views (A, B, and C) and superior views (D, E, and F) of the developing lateral roof, lateral wall, and the braincase floor of the chondrocranium and their association with developing bones. Miniaturized views at far left show location of these structures within the developing head (lateral view at top; superior view at bottom). Only the left side is fully segmented and the otic and nasal capsules are not included. Using the eMOSS staging system (Musy et al., 2018), this specimen is staged at 372 ± 2 hours (15.5 days) post conception, a developmental age that closely matches its harvesting age. Lateral (A) and superior (D) views of the chondrocranium and lateral (C) and superior (F) views of early formation of the frontal, parietal, basioccipital, and exoccipital bones were segmented from the same mouse. Lateral (B) and superior (E) views of early development of the frontal, parietal, basioccipital, and lateral occipital bones manually superimposed on their associated aspects of the chondrocranium to show these associations. The exoccipital is visible in B but hidden from view in E due to the thickness of the cartilage. Abbreviations: AO, ala orbitalis; COP, orbitoparietal commissure; CSC, sphenocochlear commissure; H, hypophyseal cartilage; O, orbital cartilage; OA, occipital arch; P, parachordal cartilage; PP, parietal plate; T, trabecular cartilage; TP, tectum posterius; TTR, tectum transversum, Y, hypochiasmatic cartilage.

Fig. 6.

Distribution of chondroblast lineage cells and osteoblast lineage cells in the ala orbitalis (chondrocranium) and frontal bone (dermatocranium) in serial sections of a mouse at E14.5 detected by immunohistochemistry using an anti-RUNX2 antibody (A, C) and an anti-COL2A1 antibody (B, D). The distribution of RUNX2 is limited to the forming frontal bone (blue arrowhead in C). COL2A1 is detected in the cartilage matrix, in the perichondrium (red arrowhead in D), and in the dorsal extension of the ala orbitalis (green arrowhead in D) that extends apically to underlie the frontal.